POSTTRANSLATIONAL MODIFICATIONS OF HUMAN INTER-ALPHA-INHIBITOR - IDENTIFICATION OF GLYCANS AND DISULFIDE BRIDGES IN HEAVY-CHAIN-1 AND HEAVY-CHAIN-2

Inter-alpha-inhibitor (I alpha I) is a serine proteinase inhibitor fou nd in high concentrations in human plasma. The protein is composed of a Light inhibitory chain called bikunin and two heavy chains of unknow n function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J, Biol. Cher n. 266, 747-751]. The aim of this study was to complete the primary st ructure by characterizing additional covalent posttranslational modifi cations of the heavy chains. Analysis revealed three N-linked oligosac charides located on Asn(251) and Asn(554) of heavy chain 1 and on Asn( 64) of heavy chain 2: all these were complex biantennary structures co mposed of (Asn)-GlcNAc(2)-Man-(Man-GlcNAc-Gal-SA)(2). In addition, the I alpha I heavy chains carried several O-linked glycans located on Th r(619) of heavy chain 1 and a cluster of four O-linked oligosaccharide s on Thr(612), Ser(619), Thr(621) and Thr(637) of heavy chain 2. The o ligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The I alpha I heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on he avy chain 1, Cys(26), appears to be modified by dihexosylation. The ot her Cys residues exclusively form intrachain disulfide bridges. In hea vy chain 1 the two disulfide bonds are formed between Cys(210) and Cys (213) and between Cys(234) and Cys(506) and in heavy chain 2, between Cys(207) and Cys(210) and between Cys(596) and Cys(597). Interestingly , three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.